The present invention provides for a novel receptor molecule, a novel molecule capable of activating the receptor, and methods of making and use thereof.
The ability of polypeptide ligands to bind cells and thereby elicit a phenotypic response such as cell growth, survival or differentiation in such cells is often mediated through receptor tyrosine kinases. The extracellular portion of each receptor tyrosine kinase (RTK) is generally the most distinctive portion of the molecule, as it provides the protein with its ligand-recognizing characteristic. Binding of a ligand to the extracellular domain results in signal transduction via an intracellular tyrosine kinase catalytic domain which transmits a biological signal to intracellular target proteins. The particular array of sequence motifs of this cytoplasmic, catalytic domain determines its access to potential kinase substrates (Mohammadi, et al., 1990, Mol. Cell. Biol., 11: 5068-5078; Fantl, et al., 1992, Cell, 69:413-413).
The tissue distribution of a particular tyrosine kinase receptor within higher organisms provides relevant data as to the biological function of the receptor. For example, the localization of a Trk family receptor, TrkB, in tissue provided some insight into the potential biological role of this receptor, as well as the ligands that bind this receptor (referred to herein as cognates). Thus, for example, in adult mice, trkB was found to be preferentially expressed in brain tissue, although significant levels of trkB mRNAs were also observed in lung, muscle, and ovaries. Further, trkB transcripts were detected in mid and late gestation embryos. In situ hybridization analysis of 14 and 18 day old mouse embryos indicated that trkB transcripts were localized in the central and peripheral nervous systems, including brain, spinal cord, spinal and cranial ganglia, paravertebral trunk of the sympathetic nervous system and various innervation pathways, suggesting that the trkB gene product may be a receptor involved in neurogenesis and early neural development as well as play a role in the adult nervous system.
The cellular environment in which an RTK is expressed may influence the biological response exhibited upon binding of a ligand to the receptor. Thus, for example, when a neuronal cell expressing a Trk receptor is exposed to a neurotrophin which binds that receptor, neuronal survival and differentiation results. When the same receptor is expressed by a fibroblast, exposure to the neurotrophin results in proliferation of the fibroblast (Glass, et al., 1991, Cell 66:405-413). Thus, it appears that the extracellular domain provides the determining factor as to the ligand specificity, and once signal transduction is initiated the cellular environment will determine the phenotypic outcome of that signal transduction.
A number of RTK families have been identified based on sequence homologies of their intracellular domains. For example, two members of the TIE (tyrosine kinase with immunoglobulin and EGF homology domains) family, known as TIE-1 and TIE-2, have 79% sequence homology in their intracellular region (Maisonpierre, et al., 1993, Oncogene 8:1631-1637). Although these receptors share similar motifs in their extracellular domain, only 32% of the sequences are identical.
A receptor having a kinase domain that is related to the Trk family has been identified in the electric ray Torpedo californica and may play a role in motor neuron induced synapses on muscle fibers. Jennings, et al. Proc. Natl. Acad. Sci. USA 90: 2895-2899 (1993). This kinase was isolated from the electric organ, a tissue which is a specialized form of skeletal muscle. The tyrosine kinase domain of this protein is related to the Trk family, while the extracellular domain is somewhat divergent from the Trks. The protein was found to be expressed at high levels in Torpedo skeletal muscle, and at much lower levels in adult Torpedo brain, spinal cord, heart, liver and testis.
Often such novel RTKs are identified and isolated by searching for additional members of known families of tyrosine kinase receptors using, for example, PCR-based screens involving known regions of homology among Trk family members. (See, for example, Maisonpierre, et al., 1993, Oncogene 8: 1631-1637). Isolation of such so called xe2x80x9corphanxe2x80x9d tyrosine kinase receptors, for which no ligand is known, and subsequent determination of the tissues in which such receptors are expressed, provides insight into the regulation of the growth, proliferation and regeneration of cells in target tissues. The identification and isolation of novel RTKs may be used as a means of identifying new ligands or activating molecules that may then be used to regulate the survival, growth, differentiation and/or regeneration of cells expressing the receptors. Further, because RTKs appear to mediate a number of important functions during development, the identification and isolation of such receptors, ligands and activating molecules enhances our understanding of developmental processes and may improve our ability to diagnose or treat abnormal conditions.
For example, the above described methods may be used to study an event that occurs during development of the neuromuscular junction (NMJ)xe2x80x94the localization of acetylcholine receptors at the synapse. It has long been known that important signals are exchanged across the NMJ (Nitkin et al., 1987, J.Cell.Biol. 105: 2471-2478; Hall, Z. W. and Sanes, J. R., 1993, Cell/Neuron (Suppl.) 72/10: 99-121; Bowe, M. A. and Fallon, J. R., 1995, Ann. Rev. Neurosci. 18: 443-462; Sanes, J. R., 1995, Devel. Biol. 6: 163-173; Burden, S. J., et al., 1995, Devel. Biol. 6: 59-65). These signals include the chemical transmitter, acetylcholine, which is released from vesicles in the nerve terminal, recognized by acetylcholine receptors (AChRs) on the muscle, and ultimately results in electrical activation and contraction of the muscle.
Muscle also provides neurotrophic factors that support survival of motor neurons (DeChiara, T. et al., 1995, Cell 83: 313-322), and the nerve may in turn provide myotrophic factors that maintain muscle mass (Helgren, M. E., et al., 1994, Cell 76: 493-504). Reciprocal signaling interactions are also critical both for the formation and maintenance of the neuromuscular junction itself. Such signals regulate recognition of nerve-to-muscle contact, arrest the growth of the incoming nerve ending, and induce formation of a highly specialized nerve terminal marked by a polarized arrangement of synaptic vesicles and active zones. Simultaneously, precisely juxtaposed with respect to the nerve terminal, a complex molecular apparatus forms on the muscle membrane. This specialized postsynaptic structure, termed the motor endplate, comprises a tiny patch on the muscle membrane which is characterized by a dense clustering of particular proteins; some of these may receive nerve-derived signals, as AChRs are known to do, while others may be involved in creating the molecular scaffold for this post-synaptic specialization.
Signals produced by the nerve induce postsynaptic clusters by at least two mechanisms. First, these signals can induce redistribution of pre-existing molecules that are initially expressed throughout the myofiber, and second, they can induce localized transcription of specific genes only by subsynaptic nuclei underlying the NMJ. Between the nerve terminal and the motor endplate is a narrow synaptic cleft containing a complex basal lamina. This basal lamina is distinguished from the adjacent extracellular matrix by the accumulation of a number of proteins, such as acetylcholinesterase and s-laminin. The synaptic basal lamina also serves as a reservoir for signaling molecules exchanged between nerve and muscle.
While the reciprocal interactions between nerve and muscle have been intensively explored for decades, many questions still remain concerning the precise nature of the signals involved in formation of the NMJ. The realization that empty sheaths of the synaptic basal lamina could induce formation of both nerve terminal specializations and motor endplates suggested that key signaling molecules might be embedded in the extracellular matrix (Sanes, J. R. et al., 1978, J.Cell. Biol. 78: 176-198; Burden, S. J., et al., 1979, J.Cell. Biol. 82: 412-425; McMahan, U. J. and Slater, C. R., 1984, J.Cell. Biol. 98: 1453-1473; Kuffler, D. P., 1986, J.Comp. Neurol. 250: 228-235). Indeed, recent findings indicate that a protein discovered for its AChR-inducing activity and thus termed ARIA (jessell, T. M., et al., 1979, PNAS (USA) 76: 5397-5401; Falls, D. L., et al., 1990, Cold Spring Harbor Symp. Quant. Biol. 55: 397-406; Falls, D. L., et al., 1993, Cell 72: 801-815) which can increase the expression of several of the AChR subunit genes (Harris, D. A., 1989, et al., Nature 337: 173-176; Martinou, J.-C., et al., 1991, PNAS (USA) 88: 7669-7673; Jo, S. A., et al., 1995, Nature 373: 158-161; Chu, G. C., et al., 1995, Neuron 14: 329-339), is localized to the synaptic basal lamina Jo, S. A., et al., 1995, Nature 373: 158-161; Goodearl, A. D., et al., 1995, J.Cell. Biol. 130: 1423-1434). Molecular cloning has revealed that ARIA corresponds to a factor alternatively referred to as neuregulin, NDF, heregulin or glia growth factor, and binds to the erbB family of RTKs (Carraway, K. L. and Burden, S. J., 1995, Curr. Opin. Neurobiol. 5: 606-612). Interestingly, neuregulin production has been demonstrated in motor neurons and neuregulin receptors, erbB3 and erbB4, have recently been localized to the motor endplate, supporting the idea that nerve-derived neuregulin provides an important signal to muscle that regulates transcription from subsynaptic nuclei (Altiok, N., et al., 1995, EMBO J. 14: 4258-4266; Moscoso, L. M., et al., 1995, Dev. Biol. 172: 158-169; Zhu, X., et al., 1995, EMBO J. 14: 5842-5848).
Another protein, known as agrin, was isolated from the synaptic basal lamina based on its ability to cause redistribution of pre-existing AChRs into clusters on the surface of cultured myotubes (Godfrey, E. W., et al., 1984, J.Cell. Biol. 99: 615-627; Rupp, F., et al., 1991, Neuron 6: 811-823; Tsim, K. W., et al., 1992, Neuron 8: 677-689). In contrast to neuregulin, agrin does not appear to regulate AChR expression. However, agrin causes the clustering of a number of synaptic components, along with AChRs, in cultured myotubes (Wallace, B. G., 1989, J.Neurosci. 9: 1294-1302).
A variety of data are consistent with the notion that agrin also acts in vivo to induce and maintain the postsynaptic membrane specialization. Most important among these are the findings that the most active forms of agrin are exclusively made by neurons and are deposited in the synaptic basal lamina (Ruegg, M. A., et al., 1992, Neuron 8: 691-699; Ferns, M., et al., 1993, Neuron 11: 491-502; Hoch, W., et al., 1993, Neuron 11: 479-490), and that antibodies to agrin block nerve-induced clustering of AChRs on cultured myotubes (Reist, N. E., et al., 1992, Neuron 8: 865-868).
The precise mechanism of action of agrin remains a mystery (Sealock, R. and Froehner, S. C., 1994, Cell 77: 617-619). Agrin is known to induce tyrosine phosphorylation of AChRs, and inhibitors of tyrosine phosphorylation block agrin-mediated clustering (Wallace, B. G., et al., 1991, Neuron 6: 869-878; Wallace, B. G., 1994, J.Cell. Biol. 125: 661-668; Qu, Z. and Huganir, R. L., 1994, J.Neurosci. 14: 6834-6841; Wallace, B. G., 1995, J.Cell. Biol. 128: 1121-1129).
Intriguing recent findings have revealed that agrin can directly bind to xcex1-dystroglycan, an extrinsic peripheral membrane protein that is attached to the cell surface by covalent linkage to xcex2-dystroglycan, which in turn couples to the intracellular cytoskeletal scaffold via an associated protein complex (Bowe, M. A., et al., 1994, Neuron 12: 1173-1180; Campanelli, J. T., et al., 1994, Cell 77: 673-674; Gee, S. H., et al., 1994, Cell 77: 675-686; Sugiyama, J., et al., 1994, Neuron 13: 103-115; Sealock, R. and Froehner, S. C., 1994, Cell 77: 617-619).
Extrasynaptically, the dystroglycan complex binds laminin on its extracellular face, and couples to the actin scaffold via a spectrin-like molecule known as dystrophin. At the synapse however, agrin (via its own laminin-like domains) may be able to substitute for laminin, whereas utrophin (a dystrophin related protein) replaces dystrophin as the link to actin (reviewed in (Bowe, M. A. and Fallon, J. R., 1995, Ann. Rev. Neurosci. 18: 443-462)). The dystroglycan complex co-clusters with AChRs in response to agrin in vitro, and components of this complex are concentrated at the endplate in vivo (reviewed in (Bowe, M. A. and Fallon, J. R., 1995, Ann. Rev. Neurosci. 18: 443-462)).
Recent evidence suggests that a 43 kD cytoplasmic protein, known as rapsyn, anchors AChRs to a sub-synaptic cytoskeleton complex, probably via interactions with the dystroglycan complex (Cartaud, J. and Changeux, J. P., 1993, Eur. J. Neurosci. 5: 191-202; Apel, E. D., et al., 1995, Neuron 15: 115-126). Gene disruption studies reveal that rapsyn is absolutely necessary for clustering of AChRs, as well as of the dystroglycan complex. However, other aspects of NMJ formation, involving presynaptic differentiation and synapse-specific transcription, are seen in mice lacking rapsyn (Gautam, M., et al., 1995, Nature 377: 232-236).
Despite the findings that agrin can bind directly to xcex1-dystroglycan, and that AChRs and the dystroglycan complex are linked and co-cluster in response to agrin, the role of dystroglycan as an agrin receptor remains unclear (Sealock, R. and Froehner, S. C., 1994, Cell 77: 617-619; Ferns, M., et al., 1996, J. Cell Biol. 132: 937-944). It has recently been reported that a 21 kD fragment of chick agrin is sufficient to induce AChR aggregation (Gesemann, M., et al., 1995, J. Cell. Biol. 128: 625-636). Dystroglycan could be directly involved in activating signaling pathways that appear to be required for clustering, such as those involving tyrosine phosphorylation, by an unknown mechanism (for example, via association with a cytoplasmic tyrosine kinase).
Alternatively, dystroglycan could be involved in couplings of agrin not only to AChRs but to a novel signaling receptor. It also remains possible that dystroglycan does not play an active or required role in initiating clustering, and is merely among an assortment of post-synaptic molecules that undergo clustering. Recent evidence indicates that the agrin fragment that is active in inducing AChR aggregation does not bind to xcex1-dystroglycan and a structural role in aggregation, rather than a signal transfer role, has been proposed for the binding of agrin to xcex1-dystroglycan (Gesemann, M., et al., 1996, Neuron 16: 755-767).
The present invention provides for a novel tyrosine kinase, termed xe2x80x9cMuSKxe2x80x9d for xe2x80x9cmuscle specific kinase,xe2x80x9d that is expressed in normal and denervated muscle, as well as other tissues including heart, spleen, ovary or retina (See Valenzuela, D., et al., 1995, Neuron 15: 573-584). The novel tyrosine kinase has alternatively been referred to as xe2x80x9cDmkxe2x80x9d for xe2x80x9cdenervated muscle kinase.xe2x80x9d (See PCT International Application No. PCT/US94/08039 published 1 February 1996 as WO 96/02643 entitled Denervated Muscle Kinase (DMK), A Receptor Of The Tyrosine Kinase Superfamily). Thus, the terms MuSK and Dmk may be used interchangeably. The protein appears to be related to the Trk family of tyrosine kinases.
The present invention further provides for an isolated nucleic acid molecule encoding MuSK.
The present invention also provides for a protein or peptide that comprises the extracellular domain of MuSK and the nucleic acid which encodes such extracellular domain. The invention further provides for vectors comprising an isolated nucleic acid molecule encoding MuSK or its extracellular domain, which can be used to express MuSK in bacteria, yeast and mammalian cells.
The present invention also provides for use of the MuSK receptor or its extracellular or intracellular domain to screen for drugs that interact with or activate MuSK. Novel agents that bind to and/or activate the receptor described herein may mediate survival, proliferation and differentiation in cells naturally expressing the receptor, but also may mediate survival, proliferation or differentiation when used to treat cells engineered to express the receptor.
In particular embodiments, the extracellular domain (soluble receptor) of MuSK is utilized in screens for cognate ligands and activating molecules. For example, the MuSK receptor activating molecule described herein may be used in a competition assay to identify agents capable of acting as receptor agonists or antagonists by competing the agents with MuSK activating molecule for phosphorylation of the MuSK receptor. Specifically, the active portion of human agrin described herein may be used as the MuSK activating molecule in a competition assay to screen for agents capable of acting as receptor agonists or antagonists.
The term xe2x80x9cMuSK activating moleculexe2x80x9d as used herein refers to a molecule which is capable of inducing phosphorylation of the MuSK receptor in the context of a differentiated muscle cell. One such activating molecule is agrin as described in the Examples set forth herein.
The present invention also provides for nucleic acid probes, capable of hybridizing with a sequence included within the nucleotide sequence encoding human MuSK or its activating molecule, useful for the detection of MuSK expressing tissue or MuSK activating molecule-expressing tissue in humans and animals. The invention further provides for antibodies capable of specifically binding MuSK or MuSK activating molecule. The antibodies may be polyclonal or monoclonal.
The present invention also has diagnostic and therapeutic utilities. In particular embodiments of the invention, methods of detecting aberrancies in the function or expression of the receptor described herein may be used in the diagnosis of muscular or other disorders. In other embodiments, manipulation of the receptor, agonists which bind this receptor, or receptor activating molecules may be used in the treatment of neurological diseases or diseases of muscle or neuromuscular unit disorders, including, but not limited to, muscular dystrophy and muscle atrophy. In further embodiments, the extracellular domain of the receptor is utilized as a blocking agent.
The present invention also provides for an isolated and purified polypeptide which activates MuSK receptor. In one embodiment, the polypeptide of the invention is encoded by a nucleotide sequence comprising the coding region of the active portion of human agrin contained in the vector designated as pBluescript human Agrin-1 (pBL-hAgrin1) that was deposited with the American Type Culture Collection (ATCC(copyright)), International Depository Authority, 10801 University Boulevard, Manassas, Va. 20110-2209 on Dec. 12, 1995 under ATCC Accession No. 97378. The present invention further provides for an isolated polypeptide which is functionally equivalent to this polypeptide.
The invention further provides for an isolated and purified nucleic acid molecule comprising a nucleotide sequence encoding the active portion of human agrin, wherein the nucleotide sequence is selected from the group consisting of:
(a) the nucleotide sequence comprising the coding region of the active portion of human agrin contained in the vector designated as pBL-hAgrin 1 (ATCC Accession No. 97378);
(b) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (a) and which encodes the active portion of human agrin; and
(c) a nucleotide sequence that, as a result of the degeneracy of the genetic code, differs from the nucleotide sequence of (a) or (b) and which encodes the active portion of human agrin.
The invention also provides for the above-described nucleic acid molecule which additionally contains a nucleotide sequence so that the encoded polypeptide contains the eight amino acids ELANEIPV at the position corresponding to amino acid position 1780 as shown in FIGS. 14A-14C.
The invention also provides for a method of promoting the growth, survival or differentiation of a MuSK receptor expressing cell comprising administering to the MuSK receptor expressing cell an effective amount of agrin or a derivative of agrin. The method may be practiced in vitro or in vivo. In one embodiment of this method, the agrin is human agrin. In another embodiment of this method, the MuSK receptor expressing cell is a cell which is normally found in the heart, spleen, ovary, retina or skeletal muscle. In another embodiment, the MuSK receptor expressing cell is a cell which has been genetically engineered to express the MuSK receptor.
The present invention also includes a method of treating a patient suffering from a muscle disease or neuromuscular disorder comprising administering to the patient an effective amount of agrin or a derivative thereof. By way of non-limiting example, the agrin may be human agrin and the derivative may be the active portion of the human agrin molecule.
The present invention also includes an antibody capable of specifically binding human agrin. More specifically, the invention includes an antibody capable of specifically binding the active portion of human agrin. The antibody may be monoclonal or polyclonal. The invention further provides a method of detecting the presence of human agrin in a sample comprising:
a) reacting the sample with an antibody capable of specifically binding human agrin under conditions whereby the antibody binds to human agrin present in the sample; and
b) detecting the bound antibody, thereby detecting the presence of human agrin in the sample.
The antibody used may be monoclonal or polyclonal. The sample may be biological tissue or body fluid. The biological tissue may be brain, muscle, or spinal cord. The body fluid may be cerebrospinal fluid, urine, saliva, blood, or a blood fraction such as serum or plasma.
The invention further provides for an isolated and purified nucleic acid molecule comprising a nucleotide sequence encoding human muscle specific kinase (MuSK) receptor, wherein the nucleotide sequence is selected from the group consisting of:
(a) the nucleotide sequence comprising the coding region of the human MuSK receptor as set forth in FIGS. 4A-4D;
(b) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (a) and which encodes a human MuSK receptor; and
(c) a nucleotide sequence that, as a result of the degeneracy of the genetic code, differs from the nucleotide sequence of (a) or (b) and which encodes a human MuSK receptor.